Method of treating COVID-19 by administering the CCR5/CCL5 (RANTES) interaction inhibitor maraviroc

ABSTRACT

Methods of treating a subject suffering from COVID-19 are provided. Aspects of the methods including administering to the subject an effective amount of an inhibitor of CCR5/CCL5 interaction, such as a CCR5 antagonist. Also provided are methods of assessing severity of a disease involving hypercytokinemia, such as COVID-19, by determining the level of CCL5/RANTES in a subject, as well as compositions for use in such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/932,636 filed Jul. 17, 2020, which application, pursuant to 35 U.S.C.§ 119 (e), claims priority to the filing dates of U.S. ProvisionalPatent Application Ser. No. 63/016,155 filed Apr. 27, 2020 and63/033,445 filed Jun. 2, 2020, the disclosures of which applications areherein incorporated by reference.

INTRODUCTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thecausative agent of coronavirus disease 2019 was first reported inDecember 2019. Since the initial cases of COVID-19 were reported fromWuhan, China in December 2019 (Huang, C. et al. Clinical features ofpatients infected with 2019 novel coronavirus in Wuhan, China. Lancet395, 497-506 (2020)), SARS-CoV-2 has emerged as a global pandemic withan ever-increasing number of severe cases requiring invasive externalventilation that threatens to overwhelm health care systems (WorldHealth Organization. Coronavirus disease (COVID-2019) situation reports.See website made up of “https://www.” before“who.int/emergencies/disease/novel-coronavirus-2019/situation-reports”).While it remains unclear why COVID-19 patients experience a spectrum ofclinical outcomes ranging from asymptomatic to severe disease, thesalient features of COVID-19 pathogenesis and mortality are rampantinflammation and CRS leading to ARDS (Mehta, P. et al. COVID-19:consider cytokine storm syndromes and immunosuppression. Lancet 395,1033-1034 (2020); Qin, C. et al. Dysregulation of immune response inpatients with COVID-19 in Wuhan, China. Clin. Infect. Dis. (2020)).Indeed, excessive immune cell infiltration into the lung, cytokinestorm, and ARDS have previously been described as defining features ofsevere disease in humans infected with the closely relatedbetacoronaviruses SARS-CoV and MERS-CoV (Channappanavar, R. & Perlman,S. Pathogenic human coronavirus infections: causes and consequences ofcytokine storm and immunopathology. Semin Immunopathol 39, 529-539(2017); Nicholls, J. M. et al. Lung pathology of fatal severe acuterespiratory syndrome. Lancet 361, 1773-1778 (2003)).

The C-C chemokine receptor 5 (CCR5) was discovered to be one of themajor HIV-1 co-receptors. Therefore, CCR5 blockade using small moleculeinhibitors or humanized monoclonal antibodies has been therapeuticallydeployed to protect certain critical immune cells populations, such asmemory CD4 T-cells and monocytes/macrophages that express CCR5, frombeing infected. Since CCR5 and its ligand CCL5 (RANTES) are alsoimportant in a host of other diverse diseases, such as non-alcoholicsteatohepatitis (NASH), graft versus host disease (GvHD), and cancer,the effects of CCR5 blockade have been extensively investigated.

In cancer, CCL5 is produced by both tumor cells but more importantly inCD3+ lymphocytes in the tumor microenvironment (TME) which are broughtin by CXCL9 and CXCL10 which are produced by CD68+ myeloid cells. CCL5causes further migration of macrophages and other immune cells into theTME where they produce anti-tumor pro-inflammatory cytokines. CCR5blockade breaks this cycle by repolarizing macrophages from a pro-tumorM2 phenotype to an anti-tumor M1 phenotype. In addition, CCR5 blockadeblocks the influx of regulatory T-cells that express CCR5 and inhibitboth the innate and cell-mediated immune responses that function both incancer immunology and viral infections.

SUMMARY

Methods of treating a subject suffering from COVID-19 are provided.Aspects of the methods including administering to the subject aneffective amount of an inhibitor of CCR5/CCL5 interaction, such as aCCR5 antagonist. Also provided are methods of assessing severity of adisease involving hypercytokinemia, such as COVID-19, by determining thelevel of CCL5/RANTES in a subject, as well as compositions for use insuch methods.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D provide the extent and phenotype of cytokines and chemokinesinvolved in hypercytokinemia in critical COVID-19. A panel of 38cytokines/chemokines was tested in plasma from ten COVID-19 patients atDay 0, Day 3 and Day 7 post-leronlimab therapy. All cytokines/chemokineswere extremely variable at Day 0 in the 10 critical patients includingthe IL-1b, IL-6, and IL-8 which are commonly associated with thecytokine storm. Conversely, CCL5/RANTES was significantly elevated inall patients. Seven days post-leronlimab, IL-6 dropped dramatically.Though CCL5/RANTES did not drop as abruptly, blockade of CCR5 byleronlimab as determined by receptor occupancy was close to 100% at day7 in all patients.

DEFINITIONS

As used herein, the term “severity of a disease” refers to the riskposed by the disease to a subject. Severity of a disease also dictatesthe extent of treatment necessary for appropriately treating thesubject. For example, a disease can be mild, moderate, severe, orcritical.

A mild disease may cause slight discomfort and may resolve without anytreatment, for example, where a subject's immune system neutralizes thedisease. A moderate disease may cause more than slight discomfort andmay require some treatment for the disease to resolve. A severe diseasecauses significant discomfort and would require extensive treatment. Acritical disease is life threatening and would require hospitalizationand extensive treatment, which may not be successful resulting in thesubject's death.

Acute respiratory distress syndrome (ARDS) is a respiratory failurecaused by rapid and widespread inflammation in the lungs. In ARDS, fluidbuilds up in the alveoli thereby preventing the lungs from filling withenough air and reduced oxygen supply to the organs.

Sepsis is a potentially life-threatening condition caused by excessiveinflammatory response to an infection. The excessive inflammatoryresponse can trigger changes that can damage multiple organ systems.

Systemic inflammatory response syndrome (SIRS) a widespread inflammatorystate affecting the entire body. Unlike sepsis, which is in response toan infection, SIRS can be in response to an infectious or noninfectiousinsult.

Toxic shock syndrome is a sudden and potentially fatal condition causedby the release of toxins from an infection, such as Staphylococcusaureus.

The term “specific binding” refers to a direct association between twomolecules, due to, for example, covalent, electrostatic, hydrophobic,and ionic and/or hydrogen-bond interactions, including interactions suchas salt bridges and water bridges. A specific binding member describes amember of a pair of molecules which have binding specificity for oneanother. The members of a specific binding pair may be naturally derivedor wholly or partially synthetically produced. One member of the pair ofmolecules has an area on its surface, or a cavity, which specificallybinds to and is therefore complementary to a particular spatial andpolar organization of the other member of the pair of molecules. Thus,the members of the pair have the property of binding specifically toeach other. Examples of pairs of specific binding members areantigen-antibody, biotin-avidin, hormone-hormone receptor,receptor-ligand, enzyme-substrate. Specific binding members of a bindingpair exhibit high affinity and binding specificity for binding with theeach other. Typically, affinity between the specific binding members ofa pair is characterized by a K_(d) (dissociation constant) of 10⁻⁶ M orless, such as 10⁻⁷ M or less, including 10⁻⁸ M or less, e.g., 10⁻⁹ M orless, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³ M orless, 10⁻¹⁴ M or less, including 10⁻¹⁵ M or less.

DETAILED DESCRIPTION

Methods of treating a subject suffering from COVID-19 are provided.Aspects of the methods including administering to the subject aneffective amount of an inhibitor of CCR5/CCL5 interaction, such as aCCR5 antagonist. Also provided are methods of assessing severity of adisease involving hypercytokinemia, such as COVID-19, by determining thelevel of CCL5/RANTES in a subject, as well as compositions for use insuch methods.

The disclosure identifies the role of the CCL5/RANTES in the severity ofa disease involving hypercytokinemia, particularly, overproduction ofimmune cells and pro-inflammatory cytokines into the lungs of a subject.The disclosure provides that, relative to controls, an increased levelof CCL5/RANTES indicates a more severe form of a disease, whereas alevel of CCL5/RANTES comparable to controls indicates a mild form of adisease. Accordingly, certain embodiments of the invention provide amethod of assessing the severity of a disease involving hypercytokinemiaby determining the level of CCL5/RANTES in a subject. Certainembodiments also provide methods of treating a disease involvinghypercytokinemia in a subject by administering an inhibitor ofCCL5/RANTES or an inhibitor of CCR5 to the subject having, relative tocontrols, an increased level of CCL5/RANTES. Further embodiments of theinvention provide a device configured to indicate whether the level ofCCL5/RANTES in a sample is above or below a predetermined threshold.Even further embodiments of the invention provide a method of treatingcoronavirus disease 2019 (COVID-19), particularly, a severe or criticalform of COVID-19, by administering to a subject an inhibitor ofCCL5/RANTES or an inhibitor of CCR5.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

Methods of Treating Hypercytokinemia Diseases, Including COVID-19

Aspects of the invention include methods of treating a subject sufferingfrom a hypercytokinemia disease, such as COVID-19. The terms “subject,”“individual,” “host,” and “patient,” are used interchangeably herein andrefer to any mammalian subject for whom diagnosis, treatment, or therapyis desired, particularly humans. By “treatment” it is meant that atleast an amelioration of one or more symptoms associated with thecondition afflicting the subject is achieved, where amelioration is usedin a broad sense to refer to at least a reduction in the magnitude of aparameter, e.g., a symptom associated with the impairment being treated.As such, treatment also includes situations where a pathologicalcondition, or at least symptoms associated therewith, are completelyinhibited, e.g., prevented from happening, or stopped, e.g., terminated,such that the adult mammal no longer suffers from the impairment, or atleast the symptoms that characterize the impairment. In some instances,“treatment”, “treating” and the like refer to obtaining a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a disease or symptomthereof and/or may be therapeutic in terms of a partial or complete curefor a disease and/or adverse effect attributable to the disease.“Treatment” may be any treatment of a disease in a mammal, and includes:(a) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving the disease, i.e., causing regression of the disease.Treatment may result in a variety of different physical manifestations,e.g., rejuvenation of tissue or organs, changes in cytokine levels, etc.Treatment of ongoing disease, where the treatment stabilizes or reducesthe undesirable clinical symptoms of the patient, occurs in someembodiments. The subject therapy may be administered prior to thesymptomatic state of the disease, during the symptomatic stage of thedisease, and in some cases after the symptomatic stage of the disease.

By hypercytokinemia disease is meant a disease condition that ischaracterized by the presence of hypercytokinemia (also known as acytokine storm). As used herein, hypercytokinemia refers to a severeimmune reaction in which the body releases too many cytokines into theblood too quickly. Cytokines play an important role in normal immuneresponses, but having a large amount of them released in the body all atonce can be harmful. Hypercytokinemia can occur as a result of aninfection, autoimmune condition, or other disease. It may also occurafter treatment with some types of immunotherapy. Signs and symptomsinclude high fever, inflammation (redness and swelling), and severefatigue and nausea. Sometimes, hypercytokinemia may be severe or lifethreatening and lead to multiple organ failure. In embodiments,hypercytokinemia diseases may be caused by a viral infection. In someinstances, the hypercytokinemia disease that is treated by methods ofthe invention is COVID-19, which is caused by SARS-CoV-2 infection. Insome instances, the disease, e.g., COVID-19, is severe or critical.

Aspects of the methods of these embodiments include administering to asubject an active agent that inhibits CCR5 mediated CCL5 signaling, suchas an inhibitor of CCR5/CCL5 binding interaction. Any convenient activeagent may be employed, where active agents include, but are not limitedto, CCR5 antagonists/inhibitors, CCL5 antagonists/inhibitors, etc.Furthermore, any convenient type of active agent may be employed, whereexamples of active agent types include, but are not limited to: smallmolecules, nucleic acids, specific binding member for CCR5 or CCL5, suchas, but not limited to, antibodies, aptamers, peptides, etc.

In some instances, the active agent is a small molecule agent thatexhibits the desired activity. Naturally occurring or synthetic smallmolecule compounds of interest include numerous chemical classes, suchas organic molecules, e.g., small organic compounds having a molecularweight of more than 50 and less than about 2,500 Daltons. Candidateagents comprise functional groups for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents may includecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Such molecules may be identified, among other ways, by employing thescreening protocols described below.

In some cases, the active agent is a protein or fragment thereof or aprotein complex. In some cases, the active agent is an antibody bindingagent or derivative thereof. The term “antibody binding agent” as usedherein includes polyclonal or monoclonal antibodies or fragments, thatare sufficient to bind to an analyte of interest, e.g., CCR5 or CCL5.The antibody fragments can be, for example, monomeric Fab fragments,monomeric Fab′ fragments, or dimeric F(ab)′2 fragments. Also within thescope of the term “antibody binding agent” are molecules produced byantibody engineering, such as single-chain antibody molecules (scFv) orhumanized or chimeric antibodies produced from monoclonal antibodies byreplacement of the constant regions of the heavy and light chains toproduce chimeric antibodies or replacement of both the constant regionsand the framework portions of the variable regions to produce humanizedantibodies. In some cases, the active agent is an enzyme or enzymecomplex. In some cases, the active includes a phosphorylating enzyme,e.g., a kinase. In some cases, the active is a complex including a guideRNA and a CRISPR effector protein, e.g., Cas9, used for targetedcleavage of a nucleic acid.

In some cases, the active agent is a nucleic acid. The nucleic acids mayinclude DNA or RNA molecules. In certain embodiments, the nucleic acidsmodulate, e.g., inhibit or reduce, the activity of a gene or protein,e.g., by reducing or downregulating the expression of the gene. Thenucleic acid may be a single stranded or double-stranded and may includemodified or unmodified nucleotides or non-nucleotides or variousmixtures and combinations thereof. In some cases, the active agentincludes intracellular gene silencing molecules by way of RNA splicingand molecules that provide an antisense oligonucleotide effect or a RNAinterference (RNAi) effect useful for inhibiting gene function. In somecases, gene silencing molecules, such as, e.g., antisense RNA, shorttemporary RNA (stRNA), double-stranded RNA (dsRNA), small interferingRNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), tinynon-coding RNA (tncRNA), snRNA, snoRNA, and other RNAi-like small RNAconstructs, may be used to target a protein-coding as well asnon-protein-coding genes. In some case, the nucleic acids includeaptamers (e.g., spiegelmers). In some cases, the nucleic acids includeantisense compounds. In some cases, the nucleic acids include moleculeswhich may be utilized in RNA interference (RNAi) such as double strandedRNA including small interfering RNA (siRNA), locked nucleic acid (LNA)inhibitors, peptide nucleic acid (PNA) inhibitors, etc.

As described above, in some instances the active agent employed inembodiments of methods of the invention is a CCR5 targeting agent, suchas a CCR5 inhibitor/antagonist. CCR5 targeting agents are described inU.S. Patent Application Pub. Nos. 1 20180303830, 20170231991,20140109245, and 20130303512; the disclosures of which are incorporatedherein by reference in their entirety. Useful drugs targeting CCR5 may,in some instances, include CCR5 antagonists, such as but not limited toe.g., small molecule (including peptide and non-peptide small molecule)inhibitors, antibodies, and the like. Non-limiting examples of CCR5antagonists include: Maraviroc (aka Selzentry or Celsentri), INCB-9471((4,6-dimethylpyrimidin-5-yl)[4-[(3S)-4-[(1R,2R)-2-ethoxy-5-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl]methanone;orally available CCR5 antagonist; PubChem CID: 49871007), Leronlimab(PRO-140, a humanized monoclonal antibody directed against CCR5),Aplaviroc(4-(4-{[(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxy)methyl]-2,5-dioxo-1,4,9-triazaspiro[5.5]undecan-9-yl]methyl}phenoxy)benzoicacid; a potent noncompetitive allosteric antagonist of the CCR5receptor), Vicriviroc(5-{4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluoromethyl)-phenyl]ethyl]-3-methylpiperazin-1-yl]-4-methylpiperidine-1-carbonyl}-4,6-dimethylpyrimidine;CCR5 entry inhibitor previously named SCH 417690 and SCH-D), fully humanmonoclonal antibodies to CCR5 (such as the HGS004 as described byLalezari et al., J Infect Dis. (2008) 197(5):721-7), and the like.

In some instances, the active agent is an antibody that binds to CCR5.An antibody that specifically binds to CCR5 can be polyclonal ormonoclonal antibody or fragments that are sufficient to bind CCR5. Theantibody fragments can be, for example, monomeric Fab fragments,monomeric Fab′ fragments, or dimeric F(ab)′₂ fragments, single-chainantibody molecules (scFv) or humanized or chimeric antibodies producedfrom monoclonal antibodies by replacement of the constant regions of theheavy and light chains to produce chimeric antibodies or replacement ofboth the constant regions and the framework portions of the variableregions to produce humanized antibodies. An antibody that specificallybinds to CCR5 can be a humanized monoclonal antibody, such as Leronlimab(PRO 40), PA14, 2D7, RoAb13, RoAb14, 45523. Certain examples of CCR5antibodies are described by the reference Olson et al. Curr Opin HIVAIDS, 2009 March; 4(2):104-111, which is herein incorporated byreference in its entirety. In some instances, the active agent isLeronlimab. In some instances, the active agent is a small moleculeinhibitor of CCR5, such as but not limited to: Maraviroc, vicriviroc,aplaviroc, SCH-C, or TAK-779.

In some embodiments, the active agent targets CCL5/RANTES. In certainembodiments, an inhibitor of CCL5/RANTES is employed as the activeagents, where inhibitors of interest include, but are not limited to:small molecules, nucleic acids, specific binding members forCCL5/RANTES, such as an antibody, aptamer, or a peptide, etc., such asdescribed above. An example of an inhibitor of CCL5/RANTES is Met-CCL5.Further examples of RANTES/CCL5 inhibitors that may be employed inembodiments of the invention include, but are not limited to, thosedescribed in United States Published Application Publication Nos.20050220790, 20060165650, 20090148455, 20120077733, 20120201826,20140377278, and 20150079099, the disclosures of which are hereinincorporated by reference.

Where desired, active agents may be administered to a subject as apharmaceutical composition. Pharmaceutical compositions formulated fortopical administration may include ointments, lotions, creams, gels,drops, sprays, liquids, salves, sticks, soaps, aerosols, and powders.Any conventional pharmaceutical excipient, such as carriers, aqueous,powder or oily bases, thickeners and the like may be used. Ointments andcreams may, for example, be formulated with an aqueous or oily base withthe addition of suitable thickening and/or gelling agents. Lotions maybe formulated with an aqueous or oily base and will, in general, alsocontain one or more emulsifying, dispersing, suspending, thickening orcoloring agents. Powders may be formed with the aid of any suitablepowder base. Drops may be formulated with an aqueous or non-aqueous basealso comprising one or more dispersing, solubilizing or suspendingagents. Aerosol sprays are conveniently delivered from pressurizedpacks, with the use of a suitable propellant.

In practicing embodiments of the invention, active agent compositionsmay be administered according to any desired dosage, such as once perday, a few or several times per day, or even multiple times per day,depending upon, among other things, the indication being treated and thejudgment of the prescribing physician. For example, in some instances,compositions that include one or more active agents may be administeredonce per day, a few or several times per day, or even multiple times perday, depending upon, among other things, the indication being treatedand the judgment of the prescribing physician.

Depending on whether systemic and/or local treatment is chosen, methodsof administration may be chosen depending also on the condition beingtreated and the pharmaceutical composition being administered.Administration of an effective amount (in one or multiple doses) of thesubject agent(s) can be done in a variety of ways, including, but notlimited to, subcutaneously, intravenously, intraperitoneally,intramuscularly, and direct injection to specified organs or tumors,systemic administration, etc. Administration of the pharmaceuticalcompositions may be through a single route or concurrently by severalroutes. As such, the active agent can be administered to a subject via asuitable route of administration and include oral, mucosal (e.g., nasal,sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous,intravenous, bolus injection, intramuscular, or intra-arterial,intraperitoneal), or transdermal.

As described above, in some instances that active agent is an anti-CCR5antibody, such as Leronlimab (PRO 140). In such embodiments, theantibody active agent is administered with a pharmaceutically acceptablecarrier. Such pharmaceutically acceptable carriers may include but arenot limited to aqueous or non-aqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, saline, andbuffered media. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, orfixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, chelatingagents, inert gases, and the like.

The dose of the composition of the invention will vary depending on thesubject and upon the particular route of administration used. Dosagescan range from 0.1 g/kg to 100,000 g/kg. In another embodiment theantibody or binding fragment thereof is formulated to deliver between100 mg/mL to 200 mg/mL of the antibody or binding fragment thereof tothe subject. In another embodiment the antibody or binding fragmentthereof is formulated to deliver between 100 mg/mL to 150 mg/mL of theantibody or binding fragment thereof to the subject. In anotherembodiment the antibody or binding fragment thereof is formulated todeliver between 150 mg/mL to 200 mg/mL of the antibody or bindingfragment thereof to the subject. In another embodiment the antibody orbinding fragment thereof is formulated to deliver 175 mg/mL of theantibody or binding fragment thereof to the subject. Based upon thecomposition, the dose can be delivered continuously, such as bycontinuous pump, or at periodic intervals, e.g., on one or more separateoccasions. Desired time intervals of multiple doses of a particularcomposition can be determined without undue experimentation by oneskilled in the art.

In one embodiment of the instant methods, the antibody or bindingfragment thereof is administered to the subject a plurality of times,and each administration delivers from 0.01 mg per kg body weight to 50mg per kg body weight of the antibody or binding fragment thereof to thesubject. In another embodiment, each administration delivers from 0.05mg per kg body weight to 25 mg per kg body weight of the antibody orbinding fragment thereof to the subject. In a further embodiment, eachadministration delivers from 0.1 mg per kg body weight to 10 mg per kgbody weight of the antibody or binding fragment thereof to the subject.In a still further embodiment, each administration delivers from 0.5 mgper kg body weight to 5 mg per kg body weight of the antibody or bindingfragment thereof to the subject. In another embodiment, eachadministration delivers from 1 mg per kg body weight to 3 mg per kg bodyweight of the antibody or binding fragment thereof to the subject. Inanother embodiment, each administration delivers about 2 mg per kg bodyweight of the antibody or binding fragment thereof to the subject.

In an embodiment, the antibody or binding fragment thereof isadministered in a formulation comprising 175 mg/mL of the anti-CCR5binding agent and may be delivered in two 1 mL shots for administrationof about 350 mg in total to a subject in need thereof.

In one embodiment, the antibody or binding fragment thereof isadministered a plurality of times, and a first administration isseparated from the subsequent administration by an interval of less thanone week. In another embodiment, the first administration is separatedfrom the subsequent administration by an interval of at least one week.In a further embodiment, the first administration is separated from thesubsequent administration by an interval of one week. In anotherembodiment, the first administration is separated from the subsequentadministration by an interval of two to four weeks. In anotherembodiment, the first administration is separated from the subsequentadministration by an interval of two weeks. In a further embodiment, thefirst administration is separated from the subsequent administration byan interval of four weeks. In yet another embodiment, the antibody orbinding fragment thereof is administered a plurality of times, and afirst administration is separated from the subsequent administration byan interval of at least one month.

In a further embodiment, the antibody or binding fragment thereof isadministered to the subject via intravenous infusion. In anotherembodiment, the antibody or binding fragment thereof is administered tothe subject via subcutaneous injection. In another embodiment, theantibody or binding fragment thereof is administered to the subject viaintramuscular injection.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, e.g., small molecule,antibody, nucleic acid, etc., the nature of the delivery vehicle, andthe like. Preferred dosages for a given compound are readilydeterminable by those of skill in the art by a variety of means.

In those embodiments where an effective amount of an active agent isadministered to the adult mammal, the amount or dosage is effective whenadministered for a suitable period of time so as to evidence a reductionin one or more symptoms of the target disease. In some instances, aneffective amount or dose of active agent will not only slow or halt theprogression of the disease condition but will also induce the reversalof the condition, i.e., will cause an improvement the subject'scondition. Where desired, effectiveness of treatment may be assessedusing any convenient protocol. Biochemically, by an “effective amount”or “effective dose” of active agent is meant an amount of active agentthat will inhibit, antagonize, decrease, reduce, or suppress by about20% or more, e.g., by 30% or more, by 40% or more, or by 50% or more, insome instances by 60% or more, by 70% or more, by 80% or more, or by 90%or more, in some cases by about 100%, i.e., to negligible amounts, andin some instances reverse, one or more target symptoms of the diseasecondition.

In certain embodiments, methods of treating a subject for ahypercytokinemia disease, e.g., Covid-19, include methods of assessingthe severity of a disease involving hypercytokinemia, such as methods ofassessing as described below.

Methods of Assessing a Disease Involving Hypercytokinemia

As summarized above, certain embodiments of the invention provide amethod of assessing the severity of a disease involving hypercytokinemiain a subject, the method comprising:

(a) determining the level of CCL5/RANTES in a test sample obtained fromthe subject, and

(b) assessing the severity of the disease based on the determined levelof CCL5/RANTES in the test sample.

As discussed above, hypercytokinemia is a potentially fatal immunereaction and involves a positive feedback loop between cytokines andimmune cells, which causes in the body highly elevated levels of variouscytokines. Hypercytokinemia is also referenced as “cytokine storm.”Hypercytokinemia typically involves increased concentration ofcytokines, such as interferons, interleukins, chemokines,colony-stimulating factors, and tumor necrosis factors. Such immunedysregulation can be an underlying factor in mortality resulting frommany infections.

In certain embodiments, the disease involving hypercytokinemia furthercomprises an overproduction of immune cells and/or pro-inflammatorycytokines into the lungs of the subject. Hypercytokinemia may result inexcessive inflammatory response in the lungs, which typically occurs inthe infections in the lungs or other organs. Such excessive inflammatoryresponse in the lung includes infiltration into the lungs by immunecells as well as excessive secretion of proinflammatory cytokines in thelungs, which in turn attracts more immune cells into the lungs. Thus,hypercytokinemia can also involve positive-feedback loop betweenactivated cells and released cytokines.

Assessing the severity of the disease may involve comparing thedetermined level of CCL5/RANTES in the test sample with the level ofCCL5/RANTES in a control sample or a reference value.

A control sample can be obtained from one or more of the following: anindividual belonging to the same species as the subject and not havingthe disease; an individual belonging to the same species as the subjectand known to have the disease with known level of severity, for example,asymptomatic, mild, moderate, severe, or critical; or the subject priorto having the disease. If a control sample is obtained from a healthyindividual and the level of CCL5/RANTES in the test sample issignificantly higher than that of the control sample, particularly,higher than that of the control sample by: between 2 fold and 200 fold;between 10 fold and 150 fold, between 20 fold and 100 fold, or between40 fold and 60 fold, the extent of the increase in the level ofCCL5/RANTES in the test sample could be used to assess the severity ofthe disease in the subject.

For example, as provided in FIG. 1D, an increase of between at leastabout 6 fold and at least about 20 fold in the CCL5/RANTES level in atest plasma sample compared to a control sample could indicate that thesubject has a severe form of COVID-19; whereas, an increase of betweenat least about 60 fold and at least about 300 fold in the CCL5/RANTESlevel in a test plasma sample compared to a control sample couldindicate that the subject has a critical form of COVID-19.

For a particular disease involving hypercytokinemia, a person ofordinary skill in the art can determine the difference in theCCL5/RANTES level in a control sample and in a test sample thatindicates different levels of severity of the disease.

The control sample and the test sample can be obtained from the sametype of an organ or tissue. The organ or tissue can be brain, eyes,pineal gland, pituitary gland, thyroid gland, parathyroid glands,thorax, heart, lung, esophagus, thymus gland, pleura, adrenal glands,appendix, gall bladder, urinary bladder, large intestine, smallintestine, kidneys, liver, pancreas, spleen, stoma, ovaries, uterus, orskin. The control sample and the test sample can also be obtained fromthe same type of a body fluid. The body fluid can be aqueous humor,vitreous humor, bile, blood, cerebrospinal fluid, chyle, endolymph,perilymph, lymph, mucus, pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sputum, synovial fluid, blood, serum orplasma.

A reference value corresponding to the level of CCL5/RANTES may indicatethe level of CCL5/RANTES associated with severity of the disease, suchas mild, moderate, severe, or critical form of a disease. As such, areference value corresponding to level of CCL5/RANTES may represent thelevel of CCL5/RANTES in a subject who has mild, moderate, severe, orcritical form of a disease.

For example, when the disease is COVID-19, the reference value can be:200 pg/ml, 250 pg/ml; 300 pg/ml, 350 pg/ml or 400 pg/ml (which wouldindicate the absence of disease); or about: 1 ng/ml, 2 ng/ml, 3 ng/ml, 4ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml (whichwould indicate severe form of COVID-19); or: 15 ng/ml, 20 ng/ml, 25ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60ng/ml (which could indicate critical form of COVID-19). Therefore, ifthe CCL5/RANTES level in a test sample from a subject is 50 ng/ml, onecould assess that the subject is suffering from or could develop acritical form of COVID-19. On the other hand, if CCL5/RANTES level in atest sample from a subject is 8 ng/ml, one could assess that the subjectis suffering from or could develop a severe form of COVID-19. Further,if CCL5/RANTES level in a test sample from a subject is 200 pg/ml, onecould assess that the subject does not have COVID-19 or has asymptomaticor mild form of COVID-19.

A person of ordinary skill in the art can obtain appropriate referencevalues based on the disease being examined and such information can beobtained from the well-known sources in the relevant art or generatedbased on the testing conducted on a case by case basis.

If the disease is COVID-19, the disease can be asymptomatic, mild,moderate, severe, or critical. An asymptomatic form of COVID-19 does notshow any symptoms in the subject. A mild form of COVID-19 may show mildform of one or more of: tiredness, fever, cough, breathlessness aftermoderate exercise, sore throat, muscle ache, headache, and diarrhea.Mild form of COVID-19 may not require management of symptoms. A moderateform of COVID-19 may show moderate form of one or more of: tiredness,fever, cough, breathlessness after slight activity, sore throat, muscleache, headache, and diarrhea. Moderate form of COVID-19 may requiremanaging the symptoms. A severe form of COVID-19 may show of one or moreof: severe tiredness, high fever, cough, breathlessness even at rest,painful breathing, loss of appetite, loss of thirst, sore throat, muscleache, headache, diarrhea, and confusion. Severe form of COVID-19 wouldtypically require significant intervention for managing symptoms, suchas: pneumonia, hypoxemic respiratory failure, ARDS, sepsis, septicshock, cardiomyopathy, arrhythmia, acute kidney injury, andcomplications from prolonged hospitalization including secondarybacterial infections, thromboembolism, gastrointestinal bleeding, andcritical illness polyneuropathy/myopathy.

A critical form of COVID-19 may show of one or more of: severetiredness, high fever, cough, breathlessness even at rest, painfulbreathing, loss of appetite, loss of thirst, sore throat, muscle ache,headache, diarrhea, confusion, severe pneumonia, ARDS, sepsis, organfailure, coma, and death. Critical form of COVID-19 requireshospitalization for managing symptoms such as: pneumonia, ARDS, sepsis,septic shock, cardiomyopathy, arrhythmia, acute kidney injury, andcomplications from prolonged hospitalization including secondarybacterial infections, thromboembolism, gastrointestinal bleeding, andcritical illness polyneuropathy/myopathy. Ventilator assisted breathingmay be required.

A disease involving hypercytokinemia can be an inflammatory disease oran infection. An inflammatory disease can be an autoimmune disease,graft rejection, multiple sclerosis, pancreatitis, or multiple organdysfunction syndrome. A disease involving hypercytokinemia can also bean infection, such as a viral, bacterial, fungal, or parasiticinfection. Additional examples of infections that can cause a diseaseinvolving hypercytokinemia are known in the art and severity of suchdiseases can be assessed according to the methods disclosed herein. Abacterial infection can comprise bacteremia, bacterial sepsis,pneumonia, cellulitis, meningitis, erysipelas, infective endocarditis,necrotizing fasciitis, prostatitis, pseudomembranous colitis,pyelonephritis, or septic arthritis. A bacterial infection can be causedby a Streptococcus spp., Staphylococcus spp., Salmonella spp.,Pseudomonas spp., Clostridium spp., Vibrio spp., Mycobacterium spp. orHaemophilus spp. Additional examples of bacteria that can cause adisease involving hypercytokinemia are known in the art and severity ofsuch bacterial infections can be assessed according to the methodsdisclosed herein. A viral infection can be caused by a coronavirus,influenza virus, Epstein-Barr virus, Human Immunodeficiency Virus, Ebolavirus, retrovirus, or variola virus. The coronavirus can be severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2). Additional examples ofviruses that can cause a disease involving hypercytokinemia are known inthe art and severity of such viral infections can be assessed accordingto the methods disclosed herein. A disease involving hypercytokinemiacan also comprise ARDS, sepsis, SIRS, or toxic shock syndrome.

Any convenient method of determining CCL5/RANTES in a same may beemployed, where various methods of determining CCL5/RANTES in a sampleare known in the art and can be used in the methods disclosed herein.Certain such methods include flow cytometry, mass spectrometry, proteinarray analysis, Western blot analysis, enzyme-linked immunosorbent assay(ELISA), or radio-immune assay (RIA).

In certain embodiments, determining the level of CCL5/RANTES isperformed by flow cytometry. Flow cytometry is a methodology usingmulti-parameter data for identifying and distinguishing betweendifferent particle (e.g., bead) types i.e., particles that vary from oneanother in terms of label (wavelength, intensity), size, etc., in afluid medium. In flow cytometrically analyzing a sample, an aliquot ofthe sample is first introduced into the flow path of the flow cytometer.When in the flow path, the particles in the sample are passedsubstantially one at a time through one or more sensing regions, whereeach of the cells is exposed separately and individually to a source oflight at a single wavelength (or in some instances two or more distinctsources of light) and measurements of cellular parameters, e.g., lightscatter parameters, and/or marker parameters, e.g., fluorescentemissions, as desired, are separately recorded for each cell. The datarecorded for each cell is analyzed in real time or stored in a datastorage and analysis means, such as a computer, for later analysis, asdesired.

In flow cytometry-based methods, particles, e.g., beads, are passed, insuspension, substantially one at a time in a flow path through one ormore sensing regions where in each region each cell is illuminated by anenergy source. The energy source may include an illuminator that emitslight of a single wavelength, such as that provided by a laser (e.g.,He/Ne or argon) or a mercury arc lamp or an LED with appropriatefilters. For example, light at 488 nm may be used as a wavelength ofemission in a flow cytometer having a single sensing region. For flowcytometers that emit light at two distinct wavelengths, additionalwavelengths of emission light may be employed, where specificwavelengths of interest include, but are not limited to: 405 nm, 535 nm,561 nm, 635 nm, 642 nm, and the like. Following excitation of a labeledspecific binding member bound to a polypeptide by an energy source, theexcited label emits fluorescence and the quantitative level of thepolypeptide on each cell may be detected, by one or more fluorescencedetectors, as it passes through the one or more sensing regions.

In flow cytometry, in addition to detecting fluorescent light emittedfrom particles labeled with fluorescent markers, detectors, e.g., lightcollectors, such as photomultiplier tubes (or “PMT”), an avalanchephotodiode (APD), etc., are also used to record light that mediated by,e.g., emitted by a label on, the particle. Flow cytometers may furtherinclude one or more electrical detectors. In certain embodiments, anelectrical detector may be employed for detecting a disturbance causedby a particle passing through an electrical field propagated across anaperture in the path of the particles. Such flow cytometers havingelectrical detectors will contain a corresponding electrical energyemitting source that propagates an electrical field across the flow pathor an aperture through which cells are directed. Any convenientelectrical field and/or combination of fields with appropriatedetector(s) may be used for the detection and/or measurement ofparticles passing through the field including but not limited to, e.g.,a direct current electrical field, alternating current electrical field,a radio-frequency field, and the like.

Flow cytometers further include data acquisition, analysis and recordingmeans, such as a computer, wherein multiple data channels record datafrom each detector for each cell as it passes through the sensingregion. The purpose of the analysis system is to classify and countcells wherein each cell presents itself as a set of digitized parametervalues and to accumulate data for the sample as a whole.

The flow cytometry can comprise a bead-based assay, such as a sandwichprotocol for bead based assay. In one embodiment, determining the levelof CCL5/RANTES by flow cytometry comprises contacting the sample with abead comprising an antibody that specifically binds to CCL5/RANTES,washing the bead and contacting the washed bead with a fluorescentlylabeled secondary antibody that specifically binds to CCL5/RANTES,washing the bead and detecting the presence of CCL5/RANTES on the beadby detecting by flow cytometry the label on the bead.

In a specific embodiment, determining the level of CCL5/RANTES by flowcytometry comprises contacting the sample with a bead comprising anantibody that specifically binds to CCL5/RANTES, washing the bead andcontacting the washed bead with a biotinylated secondary antibody thatspecifically binds to CCL5/RANTES, washing the bead and contacting thewashed bead with a fluorescently labeled streptavidin, and detecting thepresence of CCL5/RANTES on the bead by detecting by flow cytometry thefluorescent label on the bead. In some instances, the assay employed isBioLegend's LEGENDplex™ bead-based immunoassay (BioLegend, San Diego,Calif.).

The fluorescent label used to detect the bead can be selected from alarge number of dyes that are commercially available from a variety ofsources, such as Molecular Probes (Eugene, Oreg.) and Exciton (Dayton,Ohio). Examples of fluorophores of interest include, but are not limitedto, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridineand derivatives such as acridine, acridine orange, acridine yellow,acridine red, and acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); cyanine andderivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7;4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylaminocoumarin; diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein,and QFITC (XRITC); fluorescamine; IR144; IR1446; Green FluorescentProtein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™;Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate;4-methylumbeIliferone; ortho cresolphthalein; nitrotyrosine;pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin;o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G),4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolicacid and terbium chelate derivatives; xanthene; or combinations thereof.Other fluorophores or combinations thereof known to those skilled in theart may also be used, for example those available from Molecular Probes(Eugene, Oreg.) and Exciton (Dayton, Ohio).

Also of interest as specific binding members are those nucleic acid dyesor stains containing intrinsic fluorescence including those thatspecifically label DNA. Dyes and stains that are specific for DNA (orpreferentially bind double stranded polynucleotides in contrast tosingle-stranded polynucleotides) and therefore may be employed asnon-specific stains include, but are not limited to: Hoechst 33342(2′-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-1H,1′H-2,5′-bibenzimidazoletrihydrochloride) and Hoechst 33258(4-[6-(4-Methyl-1-piperazinyl)-1′,3′-dihydro-1H,2′H-2,5′-bibenzimidazol-2′-ylidene]-2,5-cyclohexadien-1-onetrihydrochloride) and others of the Hoechst series; SYTO 40, SYTO 11,12, 13, 14, 15, 16, 20, 21, 22, 23, 24, 25 (green); SYTO 17, 59 (red),DAPI, DRAQ5™ (an anthraquinone dye with high affinity for doublestranded DNA), YOYO-1, propidium iodide, YO-PRO-3, TO-PRO-3, YOYO-3 andTOTO-3, SYTOX Green, SYTOX, methyl green, acridine homodimer,7-aminoactinomycin D, 9-amino-6-chloro-2-methoxyactridine.

In certain embodiments, determining the level of CCL5/RANTES isperformed by ELISA. ELISA can be direct ELISA, indirect ELISA,competitive ELISA, or sandwich ELISA. Various methods of conductingELISA assay are known in the art and can be used in the methodsdisclosed herein. An example of such an assay is the Quantikine® ELISAHuman CCL5/RANTES Immunoassay (RnD Systems, Inc.).

Additional methods of determining plasma CCL5/RANTES level are describedin Published United States Patent Application Publication No.20120238460, the disclosure of which is herein incorporated byreference.

As such, certain embodiments of the invention provide a methodcomprising: assaying the level of CCL5/RANTES in a sample obtained froma subject suffering from a disease involving hypercytokinemia. Suchmethods can further comprise assaying the level of CCL5/RANTES in acontrol sample and/or obtaining one or more reference valuescorresponding to the level of CCL5/RANTES. The details of controlsamples and reference values discussed above are applicable to themethods of assaying CCL5/RANTES. A sample for assaying the level ofCCL5/RANTES can be obtained from a subject suffering from variousdiseases discussed above. Moreover, various methods discussed abovecould be used for assaying the level of CCL5/RANTES in a sample.

In certain embodiments, the methods of assaying the level of CCL5/RANTESin a sample obtained from a subject further comprises treating thesubject for the disease. Certain such embodiments comprise treating asubject by administering to the subject an inhibitor of CCL5/RANTES oran inhibitor of CCR5, e.g., as described above. In some embodiments,such disease is a severe or critical form of COVID-19. Varioustherapeutic methods discussed above, particularly, various inhibitors ofCCL5/RANTES or CCR5 discussed above can be used in the methods oftreating a subject discussed herein.

Devices for Assaying the Level of CCL5/Rantes

Further embodiments of the invention provide a device configured toindicate whether the level of CCL5/RANTES in a sample is above or belowa predetermined threshold.

In certain such embodiments, the device is a flow cytometer. The flowcytometer can comprise a signal processing unit that is configured toindicate whether the level of CCL5/RANTES in a sample is above or belowa predetermined threshold. Such signal processing unit can comprise aphysical computer-readable medium comprising instructions that, whenexecuted, indicate whether the level of CCL5/RANTES in a sample is aboveor below a predetermined threshold.

The predetermined threshold for CCL5/RANTES level can be about: 200pg/ml, 250 pg/ml, 300 pg/ml, 350 pg/ml, 400 pg/ml, 1 ng/ml, 2 ng/ml, 3ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml,15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50ng/ml, 55 ng/ml, or 60 ng/ml.

In certain such embodiments, the device comprises an internal controlthat provides a signal corresponding to the CCL5/RANTES level at thepredetermined threshold. Accordingly, a signal intensity higher thanthat of the internal control would indicate that the level ofCCL5/RANTES in the tested sample is higher than the predeterminedthreshold, whereas, a signal intensity lower than that of the internalcontrol would indicate that the level of CCL5/RANTES in the testedsample is lower than the predetermined threshold.

In some cases, the device is provided with a separate containercontaining a control having CCL5/RANTES at a concentration of thepredetermined threshold.

The following example(s) is/are offered by way of illustration and notby way of limitation.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, cells, andkits for methods referred to in, or related to, this disclosure areavailable from commercial vendors such as BioRad, Agilent Technologies,Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB),Takara Bio USA, Inc., and the like, as well as repositories such ase.g., Addgene, Inc., American Type Culture Collection (ATCC), and thelike.

Example 1 CCL5/RANTES Levels in Severe Cases of COVID-19

This Example demonstrates that patients with severe COVID-19 havesignificantly reduced CD8 T-effector cells and to a lesser extent CD4T-helper cells. In addition, the predominant cytokines inhypercytokinemia or cytokine storm including IL-1β, IL-8, IL-6 werequantified in patients with severe COVID-19. Also, CCL5/RANTES isupregulated in severe COVID-19 patients to an extent much greater thanthe other molecules more commonly associated with COVID-19 infection.Because CCL5/RANTES contributes to the immunopathogenesis of COVID-19lung disease and that CCL5/RANTES is involved in acute renal diseasewhich is common in these patients, blocking CCL5/RANTES can be used totreat COVID-19, particularly, a severe form of COVID-19.

1. Reduced CD8 T-cell Percentages in Critical COVID-19 Patients

To elucidate the immune status and response to infection in 10 criticalCOVID-19 patients (Table 1), routine immunophenotyping was performedfrom samples drawn at Day 0, Day 3, and Day 7. In all ten patients, CD8percentages were significantly decreased compared to normal levels,

To further determine the possible cause of this profoundimmunosuppression, high parameter flow cytometer analysis was performedusing immune subpopulation and immune function markers. These analysesdemonstrated significant immune exhaustion as evidenced by increasedPD-1 expression on CD8 and CD4 T-cells as well as increased expressionof LAG-3, and cleaved caspase 3 (CC3). Following administration of 700mg of the anti-CCR5 humanized monoclonal antibody Leronlimab, all tenpatients exhibited improvements in exhaustion markers and by Day 7 theimmune profiles resembled the normal control blood.

2. Characterization and Quantification of PlasmaCytokines/Chemokines-Predominance of CCL5

To determine the extent and phenotype of “cytokine storm” cytokines andchemokines in critical COVID-19, a panel of 38 cytokines/chemokines wasquantified in plasma from the ten critical COVID-19 patients at Day 0,Day 3 and Day 7 post-leronlimab therapy. All cytokines/chemokines wereextremely variable at Day 0 in the 10 critical patients including theIL-1b, IL-6, and IL-8 which are commonly associated with the cytokinestorm (FIGS. 1A-1C). Conversely, CCL5/RANTES was significantly elevatedin all patients (FIG. 1D). Seven days post-leronlimab, IL-6 droppeddramatically. Though RANTES did not drop as abruptly, blockade of CCR5by leronlimab as determined by receptor occupancy was close to 100% atday 7 in all patients.

Table 1 below provides representative normal cytokine levels.

TABLE 1 IL-1b IL-6 IL-8 RANTES nl 1 3.2 6.2 2.3 345 nl 2 2.8 4.9 1.4 402nl 3 2.3 3.5 1.7 322 nl 4 3 7.1 1.4 427 nl 5 1.8 4.4 2.6 383 nl 6 1.6 51.1 325 nl 7 3.4 7.8 1.9 379 nl 8 3.6 3.6 2.3 356 nl 9 1.2 4.1 1.4 413nl 10 2.8 6.3 2.5 391

Table 2 below provides cytokine levels from COVID-019 samples.

TABLE 2 COVID SAMPLES IL-1b IL-6 IL-8 RANTES 1 8.1 41.9 5.1 423 2 14.951.1 18.9 3201 3 7.7 10.5 5.7 1242 4 10.9 33.1 3.2 1234 5 6.4 5.3 4.8996 6 5.3 42.1 5.7 6608 7 4.7 13.7 6.3 2278 8 7.1 9.8 4.2 17563. Use of CCL5/RANTES in Assessing Severity of COVID-19 and Treatment

The worldwide pandemic due to COVID-19 has prompted the investigation ofnumerous therapies broadly categorized as either anti-viral agents orapproaches that temper the “Cytokine Storm.” Neither of these approachesaddresses directly the profound immunosuppression and almost all ofthese agents lack the ability to address both viral replication andimmune dysfunction. Further, approaches to quiet the cytokine storm havebeen piecemeal, inhibiting one component such as IL-6 but notsimultaneously inhibiting the other components such as IL-1b, IL-8, andGM-CSF.

The results disclosed herein show that the predominant component of theCytokine Storm, CCL5/RANTES, can be targeted using a leronlimab whichbut virtue of controlling immune trafficking of CCR5 expressing cellsand repolarization of macrophages also reduces IL-6 and restores immunehomeostasis.

The hallmark of the first reports of COVID-19 was the “cytokine storm”that leads to the lung pathology, ARDS, and in some cases death. Whileother studies are focused on the high levels of IL-6, this Exampledemonstrates that the variability in IL-6 levels was profound even inthe most critical patients. This might explain why therapeuticapproaches targeting the IL-6 receptor have had mixed success along withtoxicity in patients who already have multi-organ system failure.Conversely, profoundly increased CCL5/RANTES levels in plasma, asdemonstrated herein, may account for liver, renal toxicity but moreimportantly the mass migration and exhaustion of T-cells and macrophagesin the lungs. Treatment with leronlimab in a little as 3 dayspost-therapy begins to restore T-cell level and correct the grossalteration of CD4/CD8 ratio. CCR5 blockade can prevent the migration ofFoxP3+ t-regulatory cells (Tregs) into the tumor microenvironment.Similarly, CCR5 blockade may repolarize macrophages frompro-inflammatory cytokine including IL-6 producing M2 macrophages toanti-tumor M1 macrophages. The results disclosed herein suggest that asimilar mechanism of action is effective against COVID-19.

T-cell exhaustion was first described in mice during chronic lymphocytechoriomeningitis virus (LCMV) infection and more recently in infectionssuch as HCV and HBV as well as cancer. T-cell exhaustion though commonin chronic infections has been demonstrated to occur as a function ofantigen (viral) load (richter) and can occur in as little as 2 weeks.These data are consistent with the data presented in this reportdemonstrating persistent viral load over the first 7 days of treatment.Further evidence of T-cell exhaustion in the present study is theincreased expression of T-cell exhaustion markers PD-1 and LAG3 on bothCD4 and CD8 T-cells. Following 7 days of leronlimab, the expression ofPD-I and LAG3 significantly decreases compared to Day 0. Interestingly,correlation of PD-1 and LAG3 with liver function in these patients asreflected by decreased in liver transaminases.

CCL5/RANTES has been demonstrated to cause acute renal failure and livertoxicity, both common findings in COVID-19 infection. All 10 of thepatients in the current study has some degree of renal failure beforeadministration of leronlimab.

In summary, six out of 10 critically ill patients with COVID-19 survivedby Day 14 with 4 patients extubated and released. This compared to astudy of critically ill patients in Wuhan, China, where 31 out of 32patients died. Further, all patients exhibited immune restorationincluding T-cell numbers and improvement in exhaustion markers. Asimportant, all patients showed significantly decreased IL-6 by Day 7while CCL5/RANTES levels remained high yet blocked by close to 100% CCR5receptor occupancy at Day 7. Patients where Day 14 data was availableexhibited initiation of a drop in the level of CCL5/RANTES.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

What is claimed is:
 1. A method of treating a subject for COVID-19, themethod comprising: administering to the subject a CCR5/CCL5 interactioninhibitor to treat the subject for COVID-19, wherein the CCR5/CCL5interaction inhibitor is Maraviroc.
 2. The method according to claim 1,wherein the COVID-19 is mild, moderate, severe or critical.
 3. Themethod according to claim 1, wherein the COVID-19 is severe or critical.